Various techniques for performing quantitative amplification of a nucleic acid are known. These techniques include use of 5′ to 3′ exonuclease assays, e.g., Taqman™ probes (see, e.g., U.S. Pat. Nos. 5,210,015 and 5,487,972, Heid et al., Genome Res. 6:986-994, 1996; Holland et al., Proc. Nat'l Acad. Sci. USA 88:7276-7280, 1991; and Lee et al., Nuc. Acids Res. 21:3761-3766, 1993). Other methodologies employ one or more probe oligonucleotides that are structured such that a change in fluorescence is generated when the oligonucleotide(s) is hybridized to a target nucleic acid. For example, one such method involves a dual fluorophore approach that exploits fluorescence resonance energy transfer (FRET), e.g., LightCycler™ hybridization probes, where two oligo probes anneal to the amplicon (e.g. U.S. Pat. No. 6,174,670). The oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer. Other examples of labeled oligonucleotides that are structured to emit a signal when bound to a nucleic acid or incorporated into an extension product include: Scorpions™ probes (e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999, and U.S. Pat. No. 6,326,145), Sunrise (or Ampliflour™) primers (e.g., Nazarenko et al., Nuc. Acids Res. 25:2516-2521, 1997, and U.S. Pat. No. 6,117,635), LUX™ primers and Molecular Beacons™ probes (e.g., Tyagi et al., Nature Biotechnology 14:303-308, 1996 and U.S. Pat. No. 5,989,823).
Real-time PCR methods that are based on the use of hybridization probes have drawbacks, however, when it is desirable to use a proofreading polymerase in the amplification reaction. For example, the presence of a 3′ to 5′ exonuclease activity can lead to problems due to degradation of the probe.
Nucleic acid detection methods exist that employ error-correcting enzymes. For example, some amplification reactions rely on proofreading activity to detect differences, e.g., polymorphisms or mutations, between nucleic acid sequences (see, e.g., U.S. Pat. No. 5,391,480). In general, such an assay involves labeling the 3′ nucleotide in a primer with a fluorescent marker. The labeled oligonucleotide is hybridized to an unknown DNA sample. If the 3′ nucleotide (the query position) of the oligonucleotide is complementary to the corresponding nucleotide in the hybridized DNA, it will be insensitive to nuclease; if there is a mismatch it will be sensitive to nuclease and will be cleaved. Clipped-off fluorescent nucleotides are detected, e.g., by a decrease in fluorescence polarization (FP). In these types of assays, at least one of the primers that amplify the target is labeled.
The current invention provides a new method of quantifying an amplification reaction. The method employs a labeled hybridization probe, which is used in an amplification reaction with a polymerase and an enzyme that has 3′ to 5′ exonuclease activity, e.g., an error-correcting polymerase. The method is distinct from methods employing a 3′ to 5′ exonuclease activity that are in the prior art. For example, the methods of the present invention employ a labeled hybridization probe and unlabeled amplification primers. Further, the method is generally useful to quantify a PCR reaction, and is not limited to the detection of polymorphisms or mutations.